Induction of immune response to the 17 kDa OMPA Burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an OMP nanoemulsion-based vaccine

Abstract

Burkholderia cepacia complex (Bcc) are opportunistic bacteria associated with life-threatening illness in persons with cystic fibrosis. Once Bcc colonization is established, these antimicrobial-resistant and biofilm-forming bacteria are difficult to eradicate and are associated with increased rates of morbidity and mortality. At present, no vaccines are available to prevent the Bcc infection. There is currently a paucity of published information regarding the development of vaccines designed to prevent Burkholderia colonization. This work expands on the recent studies published by Bertot et al. [Infect Immun 75(6):2740–2752, 2007], where successful protective immune responses were generated in mice using a B. multivorans OMP-based vaccine. Here, we evaluate an experimental mucosal vaccine against Bcc using a novel mucosal adjuvant (nanoemulsion) and a novel B. cenocepacia-based OMP antigen. The OMP antigen derived from B. cenocepacia was mixed with either nanoemulsion or with PBS and delivered intranasally to CD-1 mice. Serum analysis showed robust IgG and mucosal secretory IgA immune responses in vaccinated versus control mice. The antibodies had cross-neutralizing activity against both B. cenocepacia and B. multivorans species. We found that immunized mice were protected against pulmonary colonization with B. cenocepacia. We have also identified that a 17 kDa OmpA-like protein highly conserved between Burkholderia and Ralstonia species as a new immunodominant epitope in mucosal immunization.

Introduction

Cystic fibrosis results in the functional impairment of innate respiratory defense mechanisms, providing an environment for colonization of pathogenic bacterial species such as Staphylococcus aureus and Haemophilus influenzae, and a number of opportunistic species such as Pseudomonas aeruginosa, Achromobacter xylosoxidans, Stenotrophomonas maltophilia, Ralstonia spp., Pandoraea spp., and the Burkholderia cepacia complex (Bcc) species [1]. The Bcc comprises a group of at least 17 phylogenetically related saprophytic Gram-negative bacilli, most of which can form biofilm [14]. They are particularly difficult to treat and are associated with increased rates of morbidity and mortality in CF patients. They also are among the most antimicrobial-resistant bacterial species encountered in human infections [1, 5]. Once established, the infection and associated inflammation are rarely eliminated, resulting in progressive lung disease ending in pulmonary failure and death [1, 6]. While therapeutic progress has been made in preserving functional pulmonary physiology by managing nutrition and mucosal secretions, little progress has been made in therapeutic interventions for the prevention and management of Bcc infections [1, 5]. At present, no vaccines against Bcc are available.

Because Bcc colonization occurs on the respiratory mucosa, the development of a mucosal vaccine may be of value, as this route of vaccination has been associated with both mucosal and systemic immunity [711]. Mucosal vaccine development for Bcc, though, has been limited primarily because of the lack of an identified protective antigen and the lack of effective mucosal adjuvants [7].

Outer membrane proteins (OMPs), the major surface antigens of Bcc, are considered potential protective epitopes for vaccine development. Bertot et al. [12] have previously demonstrated robust immunity and protection in an experimental infection model after nasal vaccination with OMPs from B. multivorans with adamantylamide dipeptide (AdDP) as an adjuvant and have demonstrated the potential protective value of anti-Bcc immune responses. However, as important as their contribution is, there continues to be a paucity of literature regarding Bcc vaccine development and the use of Bcc-based OMP vaccines. In these studies, we have evaluated both a novel OMP antigen derived from B. cenocepacia and a novel mucosal adjuvant (nanoemulsion). Oil-and-water nanoemulsions (NE) have previously been demonstrated by our group to be broad antimicrobials [1, 1315] as well as safe and effective non-inflammatory mucosal adjuvants for killed virus-based and recombinant protein-based vaccines [811, 16]. Here, we characterize B. cenocepacia OMPs, assess the induction of both mucosal and systemic anti-OMP antibodies using NE adjuvant, evaluate the ability of sera from mice vaccinated with B. cenocepacia to neutralize B. cenocepacia and B. multivorans, and test for protective immunity following experimental B. cenocepacia pulmonary infection.

Experimental methods

Burkholderia cenocepacia strain K56-2 stock maintenance and culture

Burkholderia cenocepacia strain K56-2 was generously provided by Dr. Pam Sokol (University of Calgary). K56-2 is a clinical isolate and has been used for Burkholderia molecular microbiology and genomic studies [17]. It is a representative of the transmissible and virulent B. cenocepacia ET12 lineage, making it a logical choice for vaccine studies [18, 19]. Burkholderia multivorans ATCC 17616 was used for cross-strain neutralization assays. Both K56-2 and ATCC 17616 strains were stored at −80°C in Luria–Bertani broth with 15% glycerol and recovered from frozen stock on brain–heart infusion agar overnight at 37°C.

Burkholderia cenocepacia OMP preparation

An overnight culture of K56-2 in brain–heart infusion media was centrifuged at 3,500 rpm for 20 min. The cell pellet was washed several times with PBS (pH 7.4), resuspended in 1 mM EDTA in PBS (pH 8.0), and then incubated for 30 min at room temperature. Post-incubation, the bacteria were passed several times through a 26-gauge needle (Becton–Dickinson) using high pressure. Cell lysis was achieved with Triton X-100 (Sigma) added to a final concentration of 2% and incubated for 10 min at room temperature. Mechanical separation was performed with multiple rounds of sonication (1 min each). The sonicated lysates were then centrifuged twice for 10 min at 6,000×g (Beckman Optima XL-100k ultracentrifuge, 25°C) with the supernatant retained after each spin. Following the second centrifugation, the supernatant was centrifuged at 100,000×g for 1 h at 4°C. The resulting pellet was resuspended in endotoxin-free PBS. To obtain the endotoxin-depleted OMP, the rough OMP preparation was purified using an endotoxin-removal column (Pierce) according to the manufacturer’s instructions. The flow-through fractions were stored at −80°C until required.

OMP analysis

The protein contained within the OMP preparation was quantified using the BCA Assay (Pierce). Western blotting and silver staining were performed according to established protocol as described previously [8]. To quantify endotoxin contaminate, OMP was analyzed using the LAL Kinetic-QCL (Lonza). Endotoxin was detected at 1.93 endotoxin units mg−1 in the endotoxin-depleted OMP preparation. DNA contaminant removal was verified by agarose-EtBr gel electrophoresis and imaged on a UV table (Fig. 1a). Oligonucleotides contaminants were not detected in either the crude OMP or the endotoxin-depleted OMP preparations.

Fig. 1
figure1

Characterization of the OMP preparation. Agarose gel electrophoresis and ethidium bromide staining (a). Lane 1, DNA ladder; Lane 2, whole cell lysate (WCL) before separation by high-speed centrifugation; Lane 3, supernatant following high-speed centrifugation; Lane 4, crude OMP preparation; Lane 5, endotoxin (ET) depleted OMP fraction. Volumetrically loaded silver stain (b) and Western blots of OMP preparation probed with serum from a mouse immunized with the endotoxin-depleted OMP-NE preparation (c) or serum from mice immunized with the OMP in PBS preparation (d). Lane A, WCL; Lane B, supernatant from the 100,000×g centrifugation; Lane C, The resuspended pellet fraction of the 100,000×g spin; Lanes D–J, endotoxin-depleted OMP fractions (the successive flow-through portions of the endotoxin column); Lanes K–N, endotoxin column retentant fractions (the successive fluid regenerated from the column after the addition of sodium deoxycholate)

OMP sequencing and verification

Sample preparation

The protein sample was separated by SDS–PAGE (Fig. 1b). The protein band immunostained with the highest intensity (~17 kDa) on Western blot (Fig. 1c) was excised and subjected to in-gel digestion. In-gel digestion was performed at the Protein Structure Facility at the University of Michigan according to procedures by Rosenfeld et al. [20] and Hellman et al. [21]. A gel slice containing 5 ρmol bovine serum albumin (BSA) was analyzed in parallel as a positive control.

Mass spectrometry

HPLC-electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was performed on a Q-tof premier mass spectrometer (Waters Inc.) fitted with a nanospray source (Waters Inc.). The mass spectrometer was calibrated with a mass accuracy within 3 ppm. On-line capillary HPLC was performed using a Waters UPLC system with an Atlantis C18 column (Waters, 100 mm inner diameter, 100 mm length, 3 mm particle size). Digests were desalted using an online trapping column (Waters, Symmetry C18, 180 mm inner diameter, 20 mm length, 5 mm particle size) before being loaded onto the Atlantis column. A data-dependent tandem mass spectrometry approach was utilized to identify peptides in which a full scan spectrum (survey scan) was used, followed by collision-induced dissociation (CID) mass spectra of all ions in the survey scan with a peak intensity rising above 20 counts per second. The survey scan was acquired in V+ mode over a mass range of 50–2,000 Da with lockmass correction (Gu-Fibrinopeptide B, [M + H]2+: 785.8426) and charge state peak selection (2+, 3+ and 4+). The MS/MS scans were acquired for 5 s with collision energy control by charge state recognition.

Data analysis and bioinformatics

Tandem mass spectra were acquired using Mass Lynx software (Version 4.1). Raw data files were first processed for lockmass correction, noise reduction, centering, and deisotoping using the Protein Lynx Global Server software Ver. 4.25 (Waters Inc.). The generated peak list files containing the fragment mass spectra were subjected to database searches using the ProteinLynxGlobal Server and Mascot (Matrix Science Inc., Boston, MA) search engines and Swiss Prot and NCBI databases, as well as the Burkholderia cenocepacia database downloaded from the Sanger Institute Web site (http://www.sanger.ac.uk).

Preparation of nanoemulsion-based OMP (OMP-NE) vaccine

NE was provided by NanoBio Corporation (Ann Arbor, MI). OMP-NE formulations were prepared by vigorously mixing the endotoxin-depleted OMP preparation (Fig. 1b, Lane F) with concentrated NE, using PBS as the diluent. For the intranasal immunizations, OMP-NEs were formulated to contain either 0.25 or 0.75 μg μl−1 OMPs mixed in 20% (v/v) NE.

Animals and immunization procedures

Pathogen-free, outbred CD-1 mice (females 6–8 weeks old) were purchased from Charles River Laboratories. The mice were housed in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care. The use of these mice was approved by the University of Michigan University Committee on Use and Care of Animals (UCUCA). The mice (n = 10 per group) were vaccinated with two administrations of either OMP-NE vaccines 4 weeks apart. Intranasal (i.n.) immunizations were performed in mice anaesthetized with isoflurane using the IMPAC anesthesia delivery system. The anesthetized animals were held in a supine position, and 20 μl (10 μl nare−1) of OMP-NE vaccine was administered slowly to the nares using a micropipette tip. Mice were immunized with either 15 μg OMP with NE, 15 μg OMP in PBS, 5 μg OMP with NE, or 5 μg OMP in PBS.

Phlebotomy, bronchoalveolar lavage, and splenocyte collection

Blood was collected from the saphenous vein every 14 days throughout the duration of the study. The terminal sample was obtained by cardiac puncture immediately following euthanasia. Whole blood samples were separated by centrifugation at 3,500 rpm (15 min) following coagulation. The serum samples were stored at −20°C until analyzed.

Bronchoalveolar lavage (BAL) fluid was obtained from the mice immediately following euthanasia as previously described [8]. In vitro measurement of the cytokine response was determined using the spleens of the vaccinated mice. Spleens were mechanically disrupted to obtain single-cell splenocyte suspension in PBS. Red blood cells were lysed with ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2 EDTA) and removed by washing the cell suspension twice in PBS. The splenocytes were then resuspended in RPMI 1640 medium supplemented with 2% FBS, 200 nM l-glutamine, and penicillin/streptomycin (100 U ml−1 and 100 mg ml−1).

Enzyme-linked immunosorbent assay

Serum IgG and mucosal secretory IgA (sIgA) were determined by enzyme-linked immunosorbent assay (ELISA) against endotoxin-depleted OMP. The OMP was prepared at 15 μg ml−1 in coating buffer (Sigma), and 100 μl was applied per well to Polysorb plates (Nunc) for a 4°C overnight incubation. Serum was serially diluted in 0.1% BSA/PBS. Either diluted serum or undiluted BAL was then added to the plates for an overnight incubation at 4°C. Following a 16-h incubation, the plates were washed and an alkaline phosphatase-conjugated anti-mouse IgG (H&L), IgG1, IgG2a, IgG2b, sIgA (a chain-specific) (Rockland Immunochemicals, Inc.) antibody was added at 1:2,000 or 1:1,000 in 0.1% BSA/PBS. The plates were read at OD405 and end titers based on naïve animal levels. Serum antibody concentrations were defined as endpoint titers (the reciprocal of the highest serum dilution producing an OD above cutoff value). The cutoff value was determined as the OD of the corresponding dilution of control sera +2 standard deviations.

Analysis of antigens recognized by serum IgG

In addition to Western blot analysis, serum lipoprotein-specific and lipopolysaccharide-specific IgG antibodies in serum were measured using ELISA. Briefly, 96-well Polysorb assay plates (Nunc) were coated with either 0.05 μg/well of crude OMP preparation, endotoxin-depleted OMP, or LPS (List Biological Laboratories). Serum was collected from mice immunized with OMP + NE 8 weeks following prime vaccination. The serum was serially diluted in 0.1% BSA/PBS and then added to the coated wells. The ELISA procedure was performed as described previously.

To evaluate the immunogenic contribution of protein and lipopolysaccharide (LPS) contained within the OMP-based vaccine, an enzymatic protein digest was performed using 250 μg of PCR-grade recombinant proteinase K (Roche) in 100 μl of either the crude OMP preparation or the endotoxin-depleted OMP preparation. Proteinase K was incubated with the OMP preparations for 16 h at room temperature. The protein digestion was verified using a silver-stained SDS–PAGE run as previously described. LPS- and OMP-protein-specific epitopes were evaluated using Western blot analysis probing with serum from mice vaccinated with either OMP-NE or OMP in PBS, or probing with an anti-Pseudomonas mallei LPS antibody (GeneTex) as previously described.

Analysis of cytokine expression

Freshly isolated mouse murine splenocytes were seeded at 4 × 106 cells ml−1 (RPMI 1640, 2% FBS) onto a 24-well plate and incubated for 72 h with OMPs (15 μg ml−1) or PHA-P mitogen (2 μg ml−1) as a control. Cell culture supernatants were collected and analyzed for the presence of cytokines using Luminex™ multi-analyte profiling beads (Luminex Corporation, Austin, TX), according to the manufacturer’s instructions.

Measurement of serum antibody neutralization

Burkholderia cenocepacia strain K56-2 and B. multivorans strain ATCC 17616 antibody-complement neutralization activities were assayed using serum harvested from mice (n = 10/group) 2 weeks following the booster vaccination administered at 4 weeks. Twenty-five microliters of serum (serially diluted 1×, 2×, 3×, 4×, or 5×) was added to 25 μl of a solution containing 1 × 103 K56-2 or B. multivorans supplementing MH medium in microculture plates (Nunc). The plates were incubated at 37°C for 48 h. Controls included 25 μl serum with 25 μl of sterile 1× PBS and 50 μl of MH medium containing 1 × 103 bacteria. Following incubation, the wells were mixed and 10 μl of solution was plated on Burkholderia selective agar (BCSA) media [22] on polystyrene culture plates (Fisherbrand) and then incubated for 72 h at 37°C prior to manual colony-forming units (CFU) enumeration.

Pulmonary bacterial challenge

The bacterial challenge studies were performed in immunized mice (n = 5 per group) or non-vaccinated control mice (n = 10) 6 weeks following the booster vaccination administered at 4 weeks using a modified version of the chronic pulmonary model of B. cepacia infection described previously [12, 23, 24]. Briefly, 5 × 107 of K56-2 suspended in 85 μl of PBS was instilled through a trans-tracheal catheter extending to the bifurcation of the main-stem bronchi in anesthetized mice. The mice were maintained for a period of 6 days following challenge. Prior to the challenge study, the mice were non-surgically implanted with programmable temperature transponders (IPTT-3000, Bio Medic Data Systems, Inc.) for non-invasive subcutaneous temperature measurement with a handheld portable scanner (DAS-6002, Bio Medic Data Systems, Inc.). In-life analysis included monitoring the clinical status of each animal by evaluating body temperature.

Immediately following euthanasia, pulmonary tissues and the spleen were collected under sterile conditions. Each organ was placed into individual containers containing 1 ml of 1× PBS (Mediatec Inc.). The tissue was then homogenized using a Tissue Tearor™ (Biospec Products Inc.) for 50 s on ice. Serial dilutions (1:102 to 1:108) of the homogenate were plated onto separated BCSA plates. All plates were then incubated for 72 h at 37°C prior to manual CFU enumeration.

Whole blood was collected in tubes containing EDTA (BD) immediately following euthanasia. Anti-coagulated blood was processed to determine total leukocytes and polymorphonuclear leukocytes by the Animal Diagnostic Laboratory at the University of Michigan, using a HEMAVETH 950 hematology analyzer (Drew Scientific, Inc.) in accordance with manufacturer’s recommendation.

Statistics

Antibody end-titer results are expressed as mean ± standard error of the mean (SEM) or ±standard deviation (SD). The statistical significance for these studies was determined by ANOVA (analysis of variance), Student t-test, and Fisher exact two-tailed test. Statistical significance for cytokine analysis was established by multivariant ANOVA. For the bacterial challenge studies, the distribution of the colonization was highly skewed, and therefore the data were transformed (natural log transformation) for data normalization. The normalized mean bacterial response in the lung and spleen was compared using an independent samples test with a two-sided P value. P < 0.05 was considered to be statistically significant. All analyses were done with 95% confidence limits. Immunization and colonization studies were repeated at least once. All in vitro assays were performed in triplicates.

Results

Identification of immunoreactive proteins

To identify immunoreactive proteins in OMPs from B. cenocepacia, mice were intranasally immunized with either 15 μg OMP in 20% NE or with 15 μg OMP in PBS. Immune sera from individual mice were used to probe electrophoretically separated and transferred OMP protein fractions. Immunoreactive OMP proteins were first identified by silver stain (Fig. 1b) followed by Western blot (Fig. 1c, d). Major reactive bands were detected at 62, 45, 17, and 10 kDa in both cases; however, the intensity of the bands was much higher with serum from mice immunized with the NE-based vaccine (Fig. 1c) when compared to the blot probed with an equivalent serum dilution from mice immunized with the OMP in PBS (Fig. 1d).

The most immune reactive moiety was approximately identified as 17 kDa protein (Fig. 1c). The 17 kDa band was isolated from the gel, and its sequence was determined by the MALDI-TOF analysis. The protein was identified as Burkholderia cepacia outer membrane lipoprotein A (OmpA) with a MW of 16.396 kDa [25, 26]. Sequence analysis using National Center for Biotechnology Information (NCBI) protein BLAST confirmed that these proteins are present in other closely related Burkholderia species (Table 1). These highly homologous proteins (87.9% sequence homology) were identified in numerous strains of Burkholderia and Ralstonia organisms, and conserved amino acid residues from the OmpA family are present in the 17 kDa protein sequence identified with OMP-NE immune sera (Table 1).

Table 1 Protein sequence alignment of OmpA-like protein family

Intranasal immunization with OMP-NE induces anti-OMP-specific antibodies

The humoral immune responses against the OMP induced by nasal vaccination with OMP were characterized in vivo in the CD-1 mice. Intranasal vaccination with either 5 or 15 μg OMP-NE formulations (OMP-NE) produced high OMP-specific IgG antibodies of 2.8 × 105 and 5.1 × 105 endpoint titers at 6 weeks, respectively, following primary vaccination (Fig. 2a). The OMPs preparations without adjuvant were also immunogenic and resulted in serum anti-OMP IgG titers of 1.9 × 104 and 3.8 × 104, respectively. However, treatment groups with NE as an adjuvant responded significantly higher (13- to 30-fold) than that of groups without adjuvant at all time points following the booster (P < 0.05). Furthermore, mice immunized with OMP in PBS have not demonstrated a significant boost effect following the second vaccination (Fig. 2a).

Fig. 2
figure2

Antibody responses against B. cenocepacia OMP. a ELISA results of the IgG response in serum post-immunization with the OMP preparation with or without nanoemulsion. Mice (n = 10/group) received a primary vaccination and a booster 4 weeks following prime. Serum anti-OMP IgG antibody concentrations are presented as mean of endpoint titers in individual sera ± SD. “*” Indicates a statistical difference (P < 0.05) in the anti-OMP IgG titers comparing OMP-NE versus OMP in PBS-immunized mice. b Mucosal antibodies sIgA and IgG against the OMP after nasal vaccine with 5 μg OMP ± NE. sIgA and IgG. Using ELISA, the levels of IgA and IgG were measured in BAL solution collected 2 weeks following booster vaccination administered at 4 weeks. BAL anti-OMP IgA and IgG are presented as mean of endpoint titers in individual sera ± SEM. The numbers on the y-axis represent the dilution factor from the starting solution which consisted of 1.6 ml of lavage fluid introduced and collected from the lungs. “*” Indicates a statistical difference (P = 0.01) in the anti-OMP IgG titers comparing OMP-NE versus OMP in PBS-immunized mice

To further characterize the OMP-NE vaccine, we evaluated its ability to elicit specific antibody production in bronchiolar secretions. Mucosal antibody production may be important for protection against Burkholderia colonization since secretory antibodies are thought to be critical effectors in protection against mucosal respiratory pathogens [27]. Mucosal immune responses were evaluated in bronchoalveolar lavage (BAL) fluid of animals immunized with 5 μg OMP-NE or 5 μg OMP in PBS. Mice vaccinated with OMP-NE had significantly higher levels of secreted IgA (P = 0.049) when compared to Naïve mice (Fig. 2b).

OMP-NE immunization yields a balanced Th1/Th2 cellular response

The analysis of the serum IgG subclass was performed to determine the T helper-type bias of the cellular response. The pattern of IgG subclass distribution shows that i.n. immunization with OMP-NE resulted in skewing of Th1-type antibodies (IgG2b) versus Th2-type IgG1 subclass antibodies (P = 0.048) (Fig. 3a). In comparison, immunization with OMP in PBS produced similar levels of IgG1 and IgG2b antibodies (Fig. 3a).

Fig. 3
figure3

Type of cellular immune responses induced by nasal OMP-NE vaccine. a Serum from mice immunized with 5 μg OMP mixed with either NE (OMP-NE) or with PBS (OMP-PBS) was analyzed for antibody subtype distribution. Mice (n = 10/group) received a prime vaccination and a booster 4 weeks following prime. The analysis was performed 2 weeks following booster vaccination administered at 4 weeks. The results are presented as ratio of the specific subclass IgG to the overall IgG titer. “*” Indicates statistical difference (P < 0.05) between IgG2b and IgG1 subtypes. The error bars represent the standard error of the IgG titers distribution. b Cytokine profiling of splenocytes of mice immunized with 5 μg mixed with either NE (OMP-NE) or with PBS (OMP-PBS). The spleens were harvested 2 weeks following booster vaccination administered at 4 weeks. Data are represented as fold change ± SEM comparing OMP-activated versus non-activated splenocytes and are normalized to responses in non-vaccinated mice. “*” Indicates statistical difference (P < 0.05) between OMP-NE and OMP in PBS groups

OMP-specific cellular responses were characterized in splenocytes obtained 6 weeks following primary vaccination with OMP-NE. The cells were stimulated with endotoxin-depleted OMP and then evaluated for specific cytokine production. In splenocytes collected from OMP-NE-vaccinated mice, Th1 cytokines IFN-γ and IL-2 production increased 22.1- and 18.6-fold, respectively, over that of splenocytes from non-vaccinated mice (P = 0.01 and 0.003, respectively) (Fig. 3b). IFN-γ was equally induced in splenocytes from mice immunized with OMP in PBS. The Th2 cytokines IL-4, IL-5, IL-6, and IL-10 were minimally induced (≤5.22-fold increase) in both OMP-NE- and OMP-immunized mice in equivalent amounts. The pattern of splenic cytokine expression and the IgG subclass distribution results suggests a balanced Th1/Th2 response to OMP-NE vaccination.

Antibody response is directed toward OMP lipoprotein and LPS

It has been previously documented that LPS is intrinsically associated with Burkholderia spp. OMP preparations [12]. To further investigate the specificity of immune response and to evaluate the possible immune stimulatory role of endotoxin in B. cencocepacia OMP preparations, we used ELISA. For this assay, plates were coated with crude OMP, endotoxin-depleted OMP or LPS and probed with sera from mice immunized with OMP-NE. The highest reactivity was detected against the endotoxin-depleted and crude OMP when compared to LPS coatings. The statistical analysis indicated a significantly higher response in endotoxin-depleted OMP versus LPS (OD405 = 2.37 ± 0.11 [P = 0.001]) and crude OMP versus LPS (OD405 = 1.49 ± 0.14 [P = 0.01]). However, the serum also contained significant levels of anti-LPS antibodies (OD405 = 0.59 ± 0.05).

Given the fact that the ELISA-based assay was not definitive and to further clarify the immunostimulatory role of endotoxins in the OMP preparation, a proteolytic proteinase K digest of both crude OMP and endotoxin-depleted OMP fractions was performed. The immunogenic epitopes were determined by Western blot analysis comparing serum antibodies generated in mice vaccinated with OMP in PBS versus from mice vaccinated with OMP-NE (Fig. 4). The results clearly indicated that antibody response was directed mainly toward 62 and 17 kDa proteins contained within the OMP preparation. This was confirmed by the complete absence of the protein bands in the proteolytically digested OMP preparations (Fig. 4—OMP-NE serum Western blot). Furthermore, when the OMP preparations were probed with commercial monoclonal anti-LPS antibody, LPS was detected as an intrinsic part of the OMP preparation. However, the proteolytic digest diminished the LPS detection. Overall, these data indicate that nasal immunization with OMP-NE enhances the production of antibodies specific against OMP proteins (Fig. 4).

Fig. 4
figure4

Identification of protein epitopes and LPS in OMP preparations. Lane 1, protein ladder; Lane 2, crude OMP preparation; Lane 3, proteinase K digested crude OMP preparation; Lane 4, endotoxin-depleted OMP; Lane 5, proteinase K digested endotoxin-depleted OMP. Western blots were probed with serum from either OMP in PBS- or OMP-NE-immunized mice or with a monoclonal anti-Pseudomonas mallei LPS antibody as indicated

Intranasal vaccination with OMP-NE results in cross-protective neutralizing serum antibody

Burkholderia cenocepacia-neutralizing antibodies were determined using a colony reduction assay. The mice intranasally vaccinated with 5 or 15 μg OMP-NE produced serum-neutralizing antibodies that inhibited 92.5 or 98.3% of B. cenocepacia colonies, respectively, when compared to serum from non-vaccinated mice (Fig. 5). The mice vaccinated with OMP without adjuvant also demonstrated a relatively high level of inhibition, resulting in 33.3 and 46.7% (for 5 and 15 μg OMP-PBS, respectively) CFU reduction. The statistical analysis showed that B. cenocepacia growth was significantly inhibited by serum derived from OMP-NE when compared to serum from those which were immunized with the OMP preparation alone (Fig. 5).

Fig. 5
figure5

Serum neutralization of B. cenocepacia or B. multivorans. Mice (n = 10/group) received a primary vaccination and a booster 4 weeks following prime with either 5 or 15 μg B. cenocepacia OMP with or without NE adjuvant. Neutralization studies were performed with serum collected at 6 weeks. The percent reductions in B. cenocepacia or B. multivorans CFU were plotted against samples with naïve serum. A statistical difference in B. cenocepacia neutralizing activity was observed between serum from mice immunized with OMP-NE and serum from mice immunized with OMP in PBS formulations (P = 9.9 × 10−5 for 5 μg OMP-NE and 0.03 for 15 μg OMP-NE). B. multivorans cross-neutralizing activity was observed between serum from mice immunized with 15 μg OMP-NE and serum from naïve mice (P = 0.04). Neutralization was not assayed using serum from 5 μg OMP-NE vaccinates. “*” Indicates a statistically significant (P < 0.05) difference in B. cenocepacia neutralizing activity between OMP-NE and OMP in PBS. “**” Indicates a statistically significant (P < 0.05) difference in B. multivorans neutralizing activity between OMP-NE-vaccinated and naïve mice

To evaluate whether the OMP-NE can produce cross-reactive immunity, serum from mice vaccinated with B. cenocepacia-derived OMP-NE was also analyzed for neutralizing activity against B. multivorans. Burkholderia multivorans was selected because it is the most common isolate cultured from CF patients infected with Bcc organisms [28]. The serum derived from mice vaccinated with OMP-NE and OMP without adjuvant inhibited B. multivorans growth by 80.1 and by 49.8%, respectively. This demonstrates induction of cross-reactive antibodies following immunization with either preparation, with a significantly higher activity produced by OMP-NE (Fig. 5).

Immunization with OMP-NE protects against pulmonary B. cenocepacia challenge and reduces incidence of sepsis

The protective effect of intranasal immunization was further tested in vivo in a lung infection model. The clearance of B. cenocepacia from pulmonary tissue was evaluated 6 days following intratracheal inoculation in mice that were intranasally immunized with the OMP-NE or the OMP without an adjuvant. Vaccination with 15 μg OMP-NE resulted in significantly higher rates of pulmonary clearance (P = 9.2 × 10−3) when compared to the non-vaccinated mice (Fig. 6a). At day 6, the average pulmonary bacterial load was 3.11 ± 2.58 CFU (represented as natural log transformation) in mice vaccinated with 15 μg OMP-NE, compared to 14.06 ± 13.75 CFU per lung in non-vaccinated mice, representing approximately a 5-log reduction in the bacterial load.

Fig. 6
figure6

Pulmonary and splenic colonization assay. Mice (n = 5/group) received a primary vaccination and a booster 4 weeks following prime with either 5 or 15 μg OMP with or without NE adjuvant. Colonization studies were performed 6 weeks following booster vaccination. a Pulmonary tissue-associated and b splenic tissue-associated CFU determined at 6 days following intratracheal challenge of 5 × 107 CFU of B. cenocepacia. Data are represented as ln (CFU/organ ± SEM). “*” Indicates a statistically significant (P < 0.05) difference in clearance of B. cenocepacia between OMP-NE-immunized and naïve mice

Splenic colonization following pulmonary inoculation with B. cenocepacia was evaluated as a means of assessing sepsis (Fig. 6b). Vaccination with 15 μg OMP-NE significantly reduced the incidence of bacteria in the spleens 6 days following the pulmonary challenge of individual mice from an average of 11.63 ± 6.98 CFU (represented as natural log transformation) per spleen in non-vaccinated mice to 0.92 ± 0.92 CFU per spleen in vaccinates (P = 0.031).

Intranasal immunization with OMP-NE attenuates systemic illness after B. cenocepacia infection

Burkholderia cenocepacia infection resulted in greater loss of thermoregulation by day 6 in the non-vaccinated mice than in the mice immunized with OMP-NE or OMP without the adjuvant. The mean body temperature 6 days following infection with B. cenocepacia was significantly lower in non-vaccinated mice (35.4°C ± 0.7) when compared to mice vaccinated with 15 μg OMP-NE (36.8°C ± 0.31, P = 0.008), 5 μg OMP-NE (37.1°C ± 0.42, P = 0.008), or 5 μg OMP without the adjuvant (36.8°C ± 0.61, P = 0.01).

The total peripheral leukocyte counts determined at the time of killing in the non-vaccinated mice were significantly higher from the values observed for the mice vaccinated with 15 μg OMP-NE (P = 0.047), 5 μg OMP-NE (P = 0.026), or 15 μg OMP in PBS (P = 0.032). The ratio of polymorphonuclear leukocytes to total peripheral leukocytes was also significantly higher in non-vaccinated mice (76.0%) compared to mice vaccinated with 15 μg OMP-NE (47.7%, P = 0.02), 5 μg OMP-NE (45.6%, P = 0.024), or 15 μg OMP in PBS (48.7%, P = 0.04) (Table 2). These data in combination with the splenic colonization results (Fig. 6b) suggest that immunization with OMP-NE resulted in decreased systemic disease following infection with B. cenocepacia.

Table 2 Polymorphonuclear leukocyte serum enumeration following challenge

Discussion

Given the morbidity associated with pulmonary infection in CF and the immune dysregulation characteristic of CF [1, 6], the development of safe and efficacious adjuvants is crucial to producing vaccines to protect against Bcc in CF patients. In this study, we demonstrated that mucosal immunization with formulations containing OMPs from B. cenocepacia mixed with NE, an experimental non-inflammatory mucosal adjuvant [8], elicited robust Bcc-specific serum and mucosal immune responses and a balanced Th1/Th2-type cellular immunity. Vaccination with OMP-NE produced antibodies neutralizing B. cenocepacia and cross-neutralizing B. multivorans species. We have also documented the efficient clearance of B. cenocepacia after experimental pulmonary infection in immunized animals.

We have identified that the immune response is associated with a 17 kDa OmpA-like protein. We have shown that the 17 kDa lipoprotein is preferentially recognized by serum antibodies from OMP-NE-vaccinated mice (Fig. 1c). This finding adds to the scarce data existing for Bcc vaccine development and suggests a rationale for the use of conserved epitopes within the 17 kDa OmpA-like proteins for induction of protective antibodies and possibly generation of vaccine effective against various strains of Burkholderia, Ralstonia and Pseudomonas pathogens. In many Gram-negative bacteria, OmpA proteins represent a major portion of outer membrane proteins and have been associated with a variety of virulence factors including adherence to and invasion of host cells, serum resistance and immune evasion, and target of immune system [29, 30]. Previously, OmpA derived from Klebsiella pneumoniae has been used to target nasal mucosa-associated dendritic cells [30] and to induce protective immune responses against respiratory syncyntial virus (RSV-A). However, there are conflicting data on the benefits of OmpA-based vaccines. For example, vaccination with Pasteurella multocida recombinant OmpA induces strong but a non-protective and deleterious Th2-type immune response in mice [30]. Despite this, the efficacy of the immune response to the OMP-NE and the protection against Bcc infection is encouraging. Current studies are underway in our laboratory utilizing purified 17 kDa OmpA for vaccination. These types of studies will be helpful in assessing the immunoprotective function of this protein.

Nasal immunization with OMP-NE produced a rapid induction of serum anti-OMP IgG, which could be subsequently boosted. In comparison with nasal immunization with AdDP adjuvant [12], the OMP-NE vaccine contained approximately threefold to tenfold lower antigen doses and after only two immunizations achieved high antibody titers and protective immunity. Mucosal as well as cell-mediated immune components are likely to be critical in the prevention of Bcc pulmonary infection, which begins with colonization of the airway epithelium and bronchial mucosa [1]. We found that immunization with either OMP-NE or OMP alone was capable of inducing antigen-specific T-cell responses and secretory antibody responses, as documented by induction of IFN-γ, IL-2, and mucosal sIgA.

Little is known about the humoral immune response to B. cepacia infection in CF patients [31]. Coinfection of CF patients with B. cepacia and P. aeruginosa has been reported to elicit anti-B. cepacia OMP IgG antibodies, which has been attributed to antigenic cross-reactivity [32, 33]. Other reports describe serum IgG and sputum sIgA antibodies to B. cepacia LPS core antigen, flagella antigens, exopolysaccharides, and elastase, among others, in infected patients [27, 3436]. However, none of these antigens are thought to be protective. Bertot et al. [12] demonstrated that the protective IgG and sIgA response in the mice was independent of LPS contamination in their preparation. The specificity of response was against 90, 72, 66, and 60 kDa proteins in that study. Our data were confirmatory for these findings. However, we also demonstrate significant response to the 17 kDa OmpA lipoprotein.

In addition to the potential utility of the OMP-NE vaccine, we demonstrated neutralization of B. cenocepacia and cross-neutralization of the B. multivorans with serum from mice vaccinated with OMP-NE. Because we identified 17 kDa OmpA as an immunodominant protein and the NCBI protein BLAST search showed that it is conserved between numerous Bcc and non-Bcc strains, it might be involved in cross-reactivity.

Novel data regarding the murine B. cenocepacia chronic lung infection model are presented in this manuscript. It is understood that animal models of B. cenocepacia infection are limited due to the contradictory reports of virulence and lack of lethality associated with pulmonary colonization [31]. Investigators have suggested that establishment of a chronic B. cenocepacia infection is best achieved with a neutropenic animal model [12, 23, 24]. In our studies, the establishment of a chronic B. cenocepacia infection was obtained by trans-tracheal instillation of 5 × 107 K56-2 organisms into healthy outbred CD-1 mice. This model was chosen in part because outbred mice may better represent the genetic diversity of human populations. Although murine CF pulmonary pathology models had been proposed, their use in vaccine studies is limited given the fact that they are often strongly associated with a strong Th2-type bias of cellular responses [37]. Although B. multivorans is isolated from the lungs of CF patients with more frequency, B. cenocepacia K56-2 was chosen for our experimental vaccine studies because it represents the transmissible and virulent ET12 lineage [19].

Immunization with OMP-NE dramatically decreased K56-2 colonization in the lungs in comparison with controls (Fig. 6a). The fact that a greater reduction was obtained in mice vaccinated with 15 μg OMP when compared to 5 μg OMP in 20% NE may suggest that cellular response mechanisms could play a critical role in clearance given the relatively small difference in serum-specific IgG response between these groups. Although reduction in the severity of systemic disease was less clear, pulmonary bacterial clearance was significantly enhanced in OMP-NE-vaccinated mice, which showed fewer B. cenocepacia organisms in the spleens when compared to non-vaccinated animals (Fig. 6b). These results suggest that the mucosal OMP-NE vaccine produced protective immunity and reduced the likelihood of sepsis, adding indirect support to the claim that increased mucosal immunity in the airways resulting from mucosal vaccination may help to prevent chronic colonization of CF patients with Bcc [12].

Although the endotoxin-depleted OMP itself was immunogenic, important differences between the OMP-NE and OMP in PBS responses were observed. For example, immunization with OMP without adjuvant produced relatively high IgG titers, they were consistently 13- to 30-fold lower than those induced by OMP-NE vaccine and have not responded to the booster immunizations (Fig. 2a). Secondly, the response induced with OMP-NE is primarily directed toward specific proteins contained within the preparation (Fig. 4). The OMP is a lipoprotein and is itself antigenic and immunostimulatory due the intrinsic endotoxin content in the protein [38]. Finally, there appears to be a difference in antigen-specific cellular response and the ability to clear pulmonary colonization following challenge in vivo in animals vaccinated with OMP-NE (Figs. 3, 6).

In summary, our study demonstrates protective immune responses generated against B. cenocepacia following mucosal vaccination with OMP-NE. OMP-NE formulations induce robust and specific humoral and cellular responses, resulting in protective immunity. By demonstrating response to a novel immunodominant 17 kDa antigenic OmpA moiety using a novel nanoemulsion adjuvant, this study expands the current body of knowledge regarding Bcc vaccine development and extends the important contribution published by Bertot et al. [12]. The 17 kDa OmpA-like protein shows potential for future vaccine development, and our study provides a rational basis for vaccines using recombinant OMP protein mixed with NE as an adjuvant.

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Acknowledgments

The authors acknowledge Dr. Henriette Remmer and staff of the MDRTC Protein Structure Facility at the University of Michigan for their expertise in sequencing of the B. cenocepacia outer membrane protein, Gayle Estep of the Human Applications Laboratory (HAL) at the University of Michigan for her work on the endotoxin content of the OMP preparation, and Kathy Welch and Heidi Reichert from the Center for Statistical Consultation and Research for their expertise in analyzing the data. This work was supported by the Carroll Haas Foundation. P. Makidon was supported by T32 RR07008 from Natl Ctr Research Resources of NIH.

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J. Knowlton and J. V. Groom II authors contributed equally as second authors.

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Makidon, P.E., Knowlton, J., Groom, J.V. et al. Induction of immune response to the 17 kDa OMPA Burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an OMP nanoemulsion-based vaccine. Med Microbiol Immunol 199, 81–92 (2010). https://doi.org/10.1007/s00430-009-0137-2

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Keywords

  • Burkholderia cepacia complex
  • OMP
  • Mucosal adjuvant
  • Vaccine
  • OmpA
  • Nanoemulsion